Thymocyte development is a remarkable process that balances the need for large numbers of peripheral T cells bearing a highly diverse repertoire with the requirement that potentially dangerous self-reactive T cells not survive. The molecular mechanisms that underlie this process are not completely understood. It is essential to learn more about thymocyte development because defects in this process underlie many human diseases, including immunodeficiency and auto-immunity, and may play a role in cancer resulting from inadequate immune surveillance. This proposal focuses on the intracellular integral membrane protein CAML (Calcium Modulating cyclophilin Ligand). We seek to learn its role and mechanism of action in T cell development and function. We originally identified the CAML gene in a two-hybrid interaction screen using cyclophilin B as bait. In the previous grant funded period, we generated CAML knockout mice, and determined that the gene is required for early embryonic development. We discovered an essential role for CAML in mediating the proliferative effects of the Epidermal Growth Factor Receptor (EGFR), and identified a direct interaction between CAML and the kinase domain of EGF-activated EGFR. We recently published new data revealing a critical role for CAML in thymocyte development. We generated CAML-conditional knockout mice and disrupted the gene specifically in developing T cells by the use of an Lck-Cre transgene. We found that loss of CAML causes a defect during thymopoiesis and essentially complete failure to produce peripheral T cells. This, together with the finding that Jurkat T leukemia cells engineered to express a dominant negative form of CAML show increased apoptosis, suggests a role for CAML in regulating T cell development, possibly through effects on survival. We have also discovered that CAML interacts with p56Lck, a protein kinase with established roles in T cell development, proliferation, and activation-induced apoptosis, and show that CAML is required for efficient p56Lck-dependent activation of the downstream signaling kinase ERK. Our central hypothesis is that CAML provides an essential function during two stages of T cell development, and that the mechanism is via enhanced resistance to apoptosis through its effects on p56Lck. To elucidate the biological mechanism of CAML in T cell development and function at the molecular, cellular, and organismal levels, we propose experiments to further investigate the precise steps in thymocyte development that are defective in the absence of the gene. In addition, we will conduct biochemical and structural experiments to determine the mechanism by which CAML regulates Lck and Erk signaling, and to discover how these enable T cells to develop and function. Project Narrative The immune system plays an essential role in protecting us from infections. It also provides an important means of blocking the development of cancer. T lymphocytes are one of the important cell types in the immune system. They have a remarkable ability to recognize a virtually unlimited number of foreign proteins, which helps them protect against infection and cancer. The mechanism T cells use to develop this ability is not yet completely understood. The experiments in this application are designed to gain important new knowledge about how T cells develop in the thymus. We will study how a protein called CAML regulates the life and death of developing T cells. We anticipate that these studies will not only help our basic understanding about the regulation of T cell death and development, but should also ultimately lead to improved treatments for diseases of the immune system, such as immunodeficiency and autoimmunity, and also for prevention of cancer.